The present invention is concerned with a process and a reagent for the determination of glycosilated hemoglobin in blood samples.
Glycosilated hemoglobin (HbA.sub.1) is formed by the non-enzymatic glycosilation of hemoglobin (HbA.sub.0). Normally, the concentration of glycosilated hemoglobin in blood is about 5%, referred to the total hemoglobin but, in the case of diabetics, this concentration may be increased 2 to 4 fold.
The determination of the glycosilated portion of the total hemoglobin has, in recent years, achieved importance in the diagnosis of diabetes (cf. L. Jovanovic and C. M. Peterson, Am. J. Med., 70, 331-338/1981). The reaction between individual hemoglobin molecules and glucose gives a stable reaction product which remains intact during the whole life time of the erythrocytes, i.e. about 100 to 200 days. Brief variations of the blood sugar content do not decisively influence the concentration of glycosilated hemoglobin. Therefore, the concentration of glycosilated hemoglobin mirrors relatively exactly the average glucose concentration in the blood of a patient over a long period of time.
Many processes are known for the determination of the glycosilated portion of the total hemoglobin. In clinical laboratories, the most widely used are chromatographic separation processes. The glycosilated portion of the hemoglobin is thereby separated from the non-glycosilated portion with the use of a chromatography column, of a microcolumn or also with the use of HPLC methods (HPLC=high pressure liquid chromatography), the columns being filled with an ion exchanger, for example with Bio Rex 70. However, all these methods are sensitive to changes of the pH value, temperature and ion concentrations. Therefore, the separation processes must be carried out very carefully in order to obtain optimum results (cf. loc. cit., page 332).
Federal Republic of Germany Patent Specification No. 29 50 457 describes a process for the determination of glycosilated hemoglobin in blood samples in which the alteration of the spectroscopic properties of a blood sample brought about by the addition of an allosteric effector is utilised for the determination of the HbA.sub.1. Only the non-glycosilated main portion of the hemoglobin is hereby influenced. In the relevant range, the measured extinction differences are very small. Furthermore, they become even smaller when the glycosilated portion of the total hemoglobin increases.
Therefore, it is an object of the present invention to provide a process with which the glycosilated hemoglobin can rapidly be determined dependably and precisely by a technically simple procedure.